Advances in molecular biochemistry and human genomics have identified numerous proteins as potential therapeutic or diagnostic agents. Compared to small molecule drugs, proteins as therapeutic agents carry the potential advantages of exerting more complicated activities, higher specificity and higher bio-compatibility. In average, protein drugs have also taken shorter time for development/approval in the past decade. However, most potential protein drugs are hampered by their pharmacokinetic profile, including short half life in circulation and insufficient penetration into target cells across biomembranes. Based on the general physicochemical properties of proteins, the reported examples of protein formulations, and the multistep processes of intracellular delivery of biomacromoles, we hypothesize that premature deactivation and insufficient penetration across lipid membranes are two key barriers of intracellular protein delivery. This hypothesis is also supported by our experiences in intracellular delivery of proteins and nucleic acids and our preliminary data on the construction and characterization of a novel protein delivery system of this proposal. This project will investigate a novel, pH-sensitiv nanometer scale protein delivery system (Lipid membrane-coated, Phosphate nano-Particles, abbreviated as LPP) that consists of a core of negatively charged calcium phosphate or magnesium phosphate nano-precipitate, and a shell of cationic lipid membranes. The cargo protein molecules will be either sandwiched between the lipid membranes of the shell or adsorbed on the inner and outer surface of the shell. In some cases, the lipid membranes will be further grafted with lipid-PEG-ligand conjugates that carry a targeting ligand at the distal end of the PEG. At pH 7.4, the delivery system is expected to protect the cargo protein and to interact with the target cells either by ionic interactions or by specific molecular recognition via the targeting ligand; after endocytosis, the elevated concentration of protons inside the endosome will dissolve the phosphate nano- core, which will then absorb water, expand in volume, and destabilize the endosomal membrane to release the cargo protein into cytosol.